Basic Helix-Loop-Helix (bHLH) transcription regulators play essential roles during mammalian neurogenesis. Vertebrate HES genes, encoding bHLH proteins, are homologues of Hairy and Enhancer of Split in Drosophila. Activated by the Notch signalling pathway, they function as negative regulators of neurogenesis by repressing the expression of proneural genes. HES-6, a new member of HES gene family, was recently cloned in the Anderson laboratory using degenerate PCR primers. Unlike other HES genes, HES-6 is transiently expressed in differentiating neuronal precursor cells in many regions of the developing central and peripheral nervous systems (CNS and PNS). Because of its unique expression pattern, I will study the functions of HES-6 gene in neurogenesis and its role in the Notch pathway as following: 1) I will generate HES-6 loss-of-function mutation in mice. The development of the CNS and PNS in homozygous HES-6 mutant embryos will be analyzed using different neuronal markers. The analyses will be emphasized on the proliferation status of neural precursors, the timing of neuronal differentiation and the expression of proneural genes. 2) I will study the functions of HES-6 using the cultured neural crest stem cells (NCSC) system. The NCSCs are multipotent cells, which can adopt different cell fates when exposed to different growth factors. I will infect the NCSC with HES-6-carrying retrovirus and analyze the effects of constitutive HES-6 expression on the cell fate determination of the NCSC. 3) I will determine the genetic interactions of HES-6 with the components of the Notch pathway by generating double mutant mice and analyzing the phenotypes.